Heparan sulfate sulfamidase (HSS) is a lysosomal exohydrolase that, when deficient, results in intralysosomal accumulation of heparan sulfate and the clinical phenotype of Sanfilippo syndrome type A. The first animal disease homolog of human Sanfilippo syndrome type A has been recently indentified in Dachshund littermates. To identify the molecular defect, the nucleotide sequences of the normal canine HSS gene and cDNA were determined using PCR-based approaches. The coding region showed 87% nucleotide homology, and 89% amino acid sequence homology, with human HSS. All exonintron borders were conserved. Sequence analysis of the entire coding region with exon-intron boundaries was performed in the propositus, a healthy littermate, and six unrelated normal dogs. Comparison revealed a 3-bp deletion, 737-739delCCA, resulting in the loss of threonine at position 246 in both alleles of the propositus and in one allele of a healthy littermate. Prediction of the three-dimensional structure of canine HSS, based on homology with human arylsulfatases A and B, suggested the pathogenic effect of this deletion. Six other sequence variations in exons, and 10 in introns, appear to be benign polymorphisms. This study supports the potential development of a canine model of Sanfilippo syndrome type A to evaluate gene therapy for this disorder. (C) 2000 Academic Press.